The solution of the human genome, and recent revelations on the role of specialized membrane microdomains in orchestrating signal transduction events, enables the application of proteomic analysis toward understanding the molecular transitions that human urinary tract cells undergo in response to disease. The focus of these studies will be on a defined regulatory cassette, the lipid raft, in developing this new knowledge. This approach should permit the rapid identification of vital regulatory elements, which by virtue of their subcellular location (near or at the plasma membrane) will potentially be useful in mechanistic studies, as therapeutic targets or markers of disease progression. By applying powerful new ICAT (isotope coded affinity tag) technology to urinary tract cells and tissues, these investigations directly address those transitional signaling events that may have the greatest chance of being of clinical value. The central hypothesis of this pilot project is that the lipid raft compartments of urinary tract smooth muscle cells (utSMC) and urothelium undergo changes in protein composition in response to stress and disease. To support this hypothesis we have two investigative aims: Aim 1) To apply ICAT proteomic technology toward the analysis of the effect of PDGF-BB on the lipid raft compartment of human bladder smooth muscle cells (SMC). In these experiments, human bladder SMC will be treated with PDGF-BB, a lipid raft-dependent smooth muscle cell mitogen. Alterations in protein expression levels in lipid raft domains of these cells will then be determined by proteomic analysis using the ICAT approach. ICAT/LC-ESI identifies and accurately determinations the relative levels of protein expression between experimental and control samples of hundreds of proteins in one experiment. These studies should permit the identification of membrane proteins involved in bladder SMC signal transduction that are potentially involved in fibroproliferative tissue remodeling. Aim 2) To demonstrate the applicability of ICAT technology to the analysis of human urinary tract disease. The major goal of this aim will be to demonstrate that the ICAT approach is suitable for the analysis of human tissue samples directly and for use in studies of genitourinary disease involving whole tissue specimens. In addition to the investigative aims, a major objective in Project D is to centralize proteomic technologies of the HURC so that the project will evolve into a Proteomics Core facility (Core 2) at the beginning of year 3 of the funding period.